A variety of chemical and biological assays exist to identify an analyte of interest in a given sample. For example, immunassays, such as enzyme-linked immunosorbent assays (ELISAs) are used in numerous diagnostic, research and screening applications. In its most common form, an ELISA detects the presence and/or concentration of an analyte in a sample using an antibody which specifically recognizes the analyte. An enzyme label, capable of providing a detectable signal, is conjugated to the antibody. The analyte is either immobilized directly onto a solid support (direct-capture ELISA) or is bound to a different specific antibody which itself is immobilized on a solid support. The presence of the immobilized analyte is detected by binding to it the detectably labeled antibody. A variety of different ELISA formats have been described. See, e.g., U.S. Pat. No. 4,011,308 to Giaever, U.S. Pat. No. 4,722,890 to Sanders et al., Re. 032696 to Schuurs et al., U.S. Pat. No. 4,016,043 to Schuurs et al., U.S. Pat. No. 3,876,504 to Koffler, U.S. Pat. No. 3,770,380 to Smith, and U.S. Pat. No. 4,372,745 to Mandle et al.
Another technique for detecting biological compounds is fluorescence in situ hybridization (FISH). Swiger et al. (1996) Environ. Mol. Mutagen. 27:245-254; Raap (1998) Mut. Res. 400:287-298; Nath et al. (1997) Biotechnic. Histol. 73:6-22. FISH allows detection of a predetermined target oligonucleotide, e.g., DNA or RNA, within a cellular or tissue preparation by, for example, microscopic visualization. Thus, FISH is an important tool in the fields of, for example, molecular cytogenetics, pathology and immunology in both clinical and research laboratories.
This method involves the fluorescent tagging of an oligonucleotide probe to detect a specific complementary DNA or RNA sequence. Specifically, FISH involves incubating an oligonucleotide probe comprising an oligonucleotide that is complementary to at least a portion of the target oligonucleotide with a cellular or tissue preparation containing or suspected of containing the target oligonucleotide. A detectable label, e.g., a fluorescent dye molecule, is bound to the oligonucleotide probe. A fluorescence signal generated at the site of hybridization is typically visualized using an epi fluorescence microscope. An alternative approach is to use an oligonucleotide probe conjugated with an antigen such as biotin or digoxygenin and a fluorescently tagged antibody directed toward that antigen to visualize the hybridization of the probe to its DNA target. A variety of FISH formats are known in the art. See, e.g., Dewald et al. (1993) Bone Marrow Transplantation 12:149-154; Ward et al. (1993) Am. J. Hum. Genet. 52:854-865; Jalal et al. (1998) Mayo Clin. Proc. 73:132-137; Zahed et al. (1992) Prenat. Diagn. 12:483-493; Kitadai et al. (1995) Clin. Cancer Res. 1:1095-1102; Neuhaus et al. (1999) Human Pathol. 30:81-86; Hack et al., eds., (1980) Association of Cytogenetic Technologists Cytogenetics Laboratory Manual. (Association of Cytogenetic Technologists, San Francisco, Calif.); Buno et al. (1998) Blood 92:2315-2321; Patterson et al. (1993) Science 260:976-979; Patterson et al. (1998) Cytometry 31:265-274; Borzi et al. (1996) J. Immunol. Meth. 193:167-176; Wachtel et al. (1998) Prenat. Diagn. 18:455-463; Bianchi (1998) J. Perinat. Med. 26:175-185; and Munne (1998) Mol. Hum. Reprod. 4:863-870.
FISH provides a powerful tool for the chromosomal localization of genes whose sequences are partially or fully known. Other applications of FISH include in situ localization of mRNA in tissues sample and localization of nongenetic DNA sequences such as telomeres.
Signal amplification is yet another method for sensitive detection of nucleic acids and other receptor/ligand interactions. Direct detection of a target nucleic acid is possible by hybridization of a complementary nucleic acid probe to the target. Detection of the complex can be achieved by numerous means, e.g., a labeled probe or a reagent dye that specifically attaches to the target/probe complex. Such "direct" detection systems are often not sensitive enough to detect a target nucleic acid in a biological sample. One method for overcoming this limitation is to employ signal amplification. Signal amplification can be done, for example, by indirectly binding multiple signal-generating molecules to an analyte through a molecule which is (1) complementary to the analyte and (2) contains multiple signal-generating molecule binding sites and which signalgenerating molecules (i) contain a detectable label, (ii) bind to or otherwise activate a label or (iii) contain sites for binding additional layers of molecules which may in turn facilitate generation of a detectable signal. Thus, rather than a single signal-generating label associated with the target molecule, signal amplification results in the association of multiple signal-generating labels associated with the target molecule and, therefore, enhanced assay sensitivity.
Nucleic acid hybridization assays are described in, for example, U.S. Pat. No. 5,681,697 to Urdea et al., U.S. Pat. No. 5,124,246 to Urdea et al., U.S. Pat. No. 4,868,105 to Urdea et al., and European Patent Publication No. 70.685, inventors Heller et al.
There are many assays designed to obtain the sequence of a DNA sample. Each of these methods shares some or all of a set of common features. These features include: sequence specificity derived from complementary oligonucleotide hybridization or annealing; a solid support or solid phase which allows separation of specifically bound assay reagents; and a label which is used for detecting the presence or absence of the specific, intended assay interaction. Examples of assays designed to detect the sequence of a DNA sample can be found in U.S. Pat. No. 5,888,731 to Yager et al., U.S. Pat. No. 5,830,711 to Barany et al., U.S. Pat. No. 5,800,994 to Martinelli et al., U.S. Pat. No. 5,792,607 to Backman et al., U.S. Pat. No. 5,716,784 to Di Cesare, U.S. Pat. No. 5,578,458 to Caskey et al., U.S. Pat. No. 5,494,810 to Barany et al., U.S. Pat. No. 4,925,785 to Wang et al., U.S. Pat. No. 4,9898,617 to Landegren et al.,
Chemical compounds are typically evaluated for potential therapeutic utility by assaying their ability to affect, for example, enzyme activity, ligand-receptor interactions, protein-protein interactions, or the like. Evaluating the effect of each individual candidate compound on a variety of systems can be tedious and time-consuming. Accordingly, protocols have been developed to evaluate rapidly multiple candidate compounds in a particular system and/or a candidate compound in a plurality of systems. Such protocols for evaluating candidate compounds have been referred to as high throughput screening (HTS).
In one typical protocol, HTS involves the dispersal of a candidate compound into a well of a multiwell cluster plate, for example, a 96-well or higher format plate, e.g., a 384-, 864-, or 1536-well plate. The effect of the compound is evaluated on the system in which it is being tested. The "throughput" of this technique, i.e., the combination of the number of candidate compounds that can be screened and the number of systems against which candidate compounds can be screened, is limited by a number of factors, including, but not limited to: only one assay can be performed per well; if conventional dye molecules are used to monitor the effect of the candidate compound, multiple excitation sources are required if multiple dye molecules are used; and as the well size becomes small (e.g., the 1536-well plate can accept about 5 .mu.l of total assay volume), consistent dispensing of individual components into a well is difficult and the amount of signal generated by each assay is significantly decreased, scaling with the volume of the assay.
A number of assay formats can be used for HTS assays. For example, the inhibitory effect of the candidate compound on, e.g., enzyme activity, ligand-receptor binding, and the like, can be measured by comparing the endpoint of the assay in the presence of a known concentration of the candidate to a reference which is performed in the absence of the candidate and/or in the presence of a known inhibitor compound. Thus, for example, a candidate compound can be identified which inhibits the binding of a ligand and its receptor, or which inhibits enzyme activity, decreasing the turnover of the enzymatic process. When this process (the inhibited process) is of clinical significance, the candidates are identified to be potential drugs for a particular condition.
A 1536-well plate is merely the physical segregation of sixteen assays within a single 96 well plate format. It would be advantageous to multiplex 16 assays into a single well of the 96 well plate. This would result in greater ease of dispensing reagents into the wells and in high signal output per well. In addition, performing multiple assays in a single well allows simultaneous determination of the potential of a candidate compound to affect a plurality of target systems. Using HTS strategies, a single candidate compound can be screened for activity as, e.g., a protease inhibitor, an inflammation inhibitor, an antiasthmatic, and the like, in a single assay.
Each of the above-described assay formats utilizes detectable labels to identify the analyte of interest. Radiolabeled molecules and compounds are frequently used to detect biological compounds both in vivo and in vitro. However, due to the inherent problems associated with the use of radioactive isotopes, nonradioactive methods of detecting biological and chemical compounds are often preferable.
For example, fluorescent molecules are commonly used as tags for detecting an analyte of interest. Fluorescence is the emission of light resulting from the absorption of radiation at one wavelength (excitation) followed by nearly immediate reradiation usually at a different wavelength (emission). Organic fluorescent dyes are typically used in this context. However, there are chemical and physical limitations to the use of such dyes. One of these limitations is the variation of excitation wavelengths of different colored dyes. As a result, the simultaneous use of two or more fluorescent tags with different excitation wavelengths requires multiple excitation light sources.
Another drawback of organic dyes is the deterioration of fluorescence intensity upon prolonged and/or repeated exposure to excitation light. This fading, called photobleaching, is dependent on the intensity of the excitation light and the duration of the illumination. In addition, conversion of the dye into a nonfluorescent species is irreversible. Furthermore, the degradation products of dyes are organic compounds which may interfere with the biological processes being examined.
Additionally, spectral overlap exists from one dye to another. This is due, in part, to the relatively wide emission spectra of organic dyes and the overlap of the spectra near the tailing region. Few low molecular weight dyes have a combination of a large Stokes shift, which is defined as the separation of the absorption and emission maxima, and high fluorescence output. In addition, low molecular weight dyes may be impractical for some applications because they do not provide a bright enough fluorescent signal.
Furthermore, the differences in the chemical properties of standard organic fluorescent dyes make multiple, parallel assays impractical as different chemical reactions may be involved for each dye used in the variety of applications of fluorescent labels.
Thus, there is a continuing need in the assay art for labels with the following features: (i) high fluorescent intensity (for detection in small quantities), (ii) adequate separation between the absorption and emission frequencies, (iii) good solubility, (iv) ability to be readily linked to other molecules, (v) stability towards harsh conditions and high temperatures, (vi) a symmetric, nearly gaussian emission lineshape for easy deconvolution of multiple colors, and (vii) compatibility with automated analysis. At present, none of the conventional fluorescent labels satisfies all of these requirements.